A pocket-factor-triggered conformational switch in the hepatitis B virus capsid.

Viral hepatitis is growing into an epidemic illness, and it is urgent to neutralize the main culprit, hepatitis B virus (HBV), a small-enveloped retrotranscribing DNA virus. An intriguing observation in HB virion morphogenesis is that capsids with immature genomes are rarely enveloped and secreted. This prompted, in 1982, the postulate that a regulated conformation switch in the capsid triggers envelopment. Using solid-state NMR, we identified a stable alternative conformation of the capsid. The structural variations focus on the hydrophobic pocket of the core protein, a hot spot in capsid-envelope interactions. This structural switch is triggered by specific, high-affinity binding of a pocket factor. The conformational change induced by the binding is reminiscent of a maturation signal. This leads us to formulate the "synergistic double interaction" hypothesis, which explains the regulation of capsid envelopment and indicates a concept for therapeutic interference with HBV envelopment.

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA.

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg2+:vanadate, while the second site is more 'open' allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31P-31P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.

Fast Magic-Angle-Spinning NMR Reveals the Evasive Hepatitis†B Virus Capsid C-Terminal Domain.

Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

Emerging Structural Understanding of Amyloid Fibrils by Solid-State NMR.

Amyloid structures at atomic resolution have remained elusive mainly because of their extensive polymorphism and because their polymeric properties have hampered structural studies by classical approaches. Progress in sample preparation, as well as solid-state NMR methods, recently enabled the determination of high-resolution 3D structures of fibrils such as the amyloid-ß fibril, which is involved in Alzheimer's disease. Notably, the simultaneous but independent structure determination of Aß1-42, a peptide that forms fibrillar deposits in the brain of Alzheimer patients, by two independent laboratories, which yielded virtually identical results, has highlighted how structures can be obtained that allow further functional investigation.

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

Paramagnetic Solid-State NMR to Localize the Metal-Ion Cofactor in an Oligomeric DnaB Helicase.

Paramagnetic metal ions can be inserted into ATP-fueled motor proteins by exchanging the diamagnetic Mg2+ cofactor with Mn2+ or Co2+ . Then, paramagnetic relaxation enhancement (PRE) or pseudo-contact shifts (PCSs) can be measured to report on the localization of the metal ion within the protein. We determine the metal position in the oligomeric bacterial DnaB helicase from Helicobacter pylori complexed with the transition-state ATP-analogue ADP:AlF4 - and single-stranded DNA using solid-state NMR and a structure-calculation protocol employing CYANA. We discuss and compare the use of Mn2+ and Co2+ in localizing the ATP cofactor in large oligomeric protein assemblies. 31 P PCSs induced in the Co2+ -containing sample are then used to localize the DNA phosphate groups on the Co2+ PCS tensor surface enabling structural insights into DNA binding to the DnaB helicase.

Dimer Organization of Membrane-Associated NS5A of Hepatitis†C Virus as Determined by Highly Sensitive 1 H-Detected Solid-State NMR.

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.

Nucleotide Binding Modes in a Motor Protein Revealed by 31 P- and 1 H-Detected MAS Solid-State NMR Spectroscopy.

Protein-nucleic acid interactions play important roles not only in energy-providing reactions, such as ATP hydrolysis, but also in reading, extending, packaging, or repairing genomes. Although they can often be analyzed in detail with X-ray crystallography, complementary methods are needed to visualize them in complexes, which are not crystalline. Here, we show how solid-state NMR spectroscopy can detect and classify protein-nucleic interactions through site-specific 1 H- and 31 P-detected spectroscopic methods. The sensitivity of 1 H chemical-shift values on noncovalent interactions involved in these molecular recognition processes is exploited allowing us to probe directly the chemical bonding state, an information, which is not directly accessible from an X-ray structure. We show that these methods can characterize interactions in easy-to-prepare sediments of the 708 kDa dodecameric DnaB helicase in complex with ADP:AlF4- :DNA, and this despite the very challenging size of the complex.

Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus.

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D 1 H-detected 1 H,15 N and 3D 1 H,13 C,15 N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.

Protein NMR Spectroscopy at 150†kHz Magic-Angle Spinning Continues To Improve Resolution and Mass Sensitivity.

Spectral resolution is the key to unleashing the structural and dynamic information contained in NMR spectra. Fast magic-angle spinning (MAS) has recently revolutionized the spectroscopy of biomolecular solids. Herein, we report a further remarkable improvement in the resolution of the spectra of four fully protonated proteins and a small drug molecule by pushing the MAS rotation frequency higher (150 kHz) than the more routinely used 100 kHz. We observed a reduction in the average homogeneous linewidth by a factor of 1.5 and a decrease in the observed linewidth by a factor 1.25. We conclude that even faster MAS is highly attractive and increases mass sensitivity at a moderate price in overall sensitivity.

In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes.

Single-stranded, positive-sense RNA viruses encode essential replication polyproteins which are composed of several domains. They are usually subjected to finely regulated proteolytic maturation processes to generate cleavage intermediates and end-products. Both polyproteins and maturation products play multiple key roles that ultimately allow synthesis of viral genome progeny. Despite the importance of these proteins in the course of viral replication, their structural properties, including the conformational changes regulating their numerous functions, are poorly described at the structural level. This lack of information is mainly due to the extreme difficulty to express large, membrane-bound, multi-domain proteins with criteria suitable for structural biology methods. To tackle this challenge, we have used a wheat-germ cell-free expression system. We firstly establish that this approach allows to synthesize viral polyproteins encoded by two unrelated positive-sense RNA viruses, a human norovirus and a plant tymovirus. Then, we demonstrate that these polyproteins are fully functional and are spontaneously auto-cleaved by their active protease domain, giving rise to natural maturation products. Moreover, we show that introduction of point mutations in polyproteins allows to inhibit the proteolytic maturation process of each virus. This allowed us to express and partially purify the uncleaved full-length norovirus polyprotein and the tymoviral RNA-dependent RNA polymerase. Thus, this study provides a powerful tool to obtain soluble viral polyproteins and their maturation products in order to conduct challenging structural biology projects and therefore solve unanswered questions.

ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.

Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.

Spinning faster: protein NMR at MAS frequencies up to 126 kHz.

We report linewidth and proton T1, T1ρ and T2' relaxation data of the model protein ubiquitin acquired at MAS frequencies up to 126 kHz. We find a predominantly linear improvement in linewidths and coherence decay times of protons with increasing spinning frequency in the range from 93 to 126 kHz. We further attempt to gain insight into the different contributions to the linewidth at fast MAS using site-specific analysis of proton relaxation parameters and present bulk relaxation times as a function of the MAS frequency. For microcrystalline fully-protonated ubiquitin, inhomogeneous contributions are only a minor part of the proton linewidth, and at 126 kHz MAS coherent effects are still dominating. We furthermore present site-specific proton relaxation rate constants during a spinlock at 126 kHz MAS, as well as MAS-dependent bulk T1ρ (1HN).

A Substantial Structural Conversion of the Native Monomer Leads to in-Register Parallel Amyloid Fibril Formation in Light-Chain Amyloidosis.

Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show ß-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially 15 N:13 C-labeled samples, that the ß-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.

Quantifying proton NMR coherent linewidth in proteins under fast MAS conditions: a second moment approach.

Proton detected solid-state NMR under fast magic-angle-spinning (MAS) conditions is currently redefining the applications of solid-state NMR, in particular in structural biology. Understanding the contributions to the spectral linewidth is thereby of paramount importance. When disregarding the sample-dependent inhomogeneous contributions, the NMR proton linewidth is defined by homogeneous broadening, which has incoherent and coherent contributions. Understanding and disentangling these different contributions in multi-spin systems like proteins is still an open issue. The coherent contribution is mainly caused by the dipolar interaction under MAS and is determined by the molecular structure and the proton chemical shifts. Numerical simulation approaches based on numerically exact direct integration of the Liouville-von Neumann equation can give valuable information about the lineshape, but are limited to small spin systems (<12 spins). We present an alternative simulation method for the coherent contributions based on the rapid and partially analytic calculation of the second moments of large spin systems. We first validate the method on a simple system by predicting the 19F linewidth in CaF2 under MAS. We compare simulation results to experimental data for microcrystalline ubiquitin (deuterated 100% back-exchanged at 110 kHz and fully-protonated at 125 kHz). Our results quantitatively explain the observed linewidth per-residue basis for the vast majority of residues.

Atomic-resolution structure of a disease-relevant Aß(1-42) amyloid fibril.

Amyloid-ß (Aß) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aß(1-40) and Aß(1-42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aß(1-42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer's disease (AD) patients. Although small Aß oligomers are believed to be the neurotoxic species, Aß amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aß(1-42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15-42 forming a double-horseshoe-like cross-ß-sheet entity with maximally buried hydrophobic side chains. Residues 1-14 are partially ordered and in a ß-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure.

Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy.

Selective isotope labeling is central in NMR experiments and often allows to push the limits on the systems investigated. It has the advantage to supply additional resolution by diminishing the number of signals in the spectra. This is particularly interesting when dealing with the large protein systems which are currently becoming accessible to solid-state NMR studies. Isotope labeled proteins for NMR experiments are most often expressed in E. coli systems, where bacteria are grown in minimal media supplemented with 15NH4Cl and 13C-glucose as sole source of nitrogen and carbon. For amino acids selective labeling or unlabeling, specific amino acids are supplemented in the minimal medium. The aim is that they will be incorporated in the protein by the bacteria. However, E. coli amino-acid anabolism and catabolism tend to interconnect different pathways, remnant of a subway system. These connections lead to inter conversion between amino acids, called scrambling. A thorough understanding of the involved pathways is thus important to obtain the desired labeling schemes, as not all combinations of amino acids are adapted. We present here a detailed overview of amino-acid metabolism in this context. Each amino-acid pathway is described in order to define accessible combinations for 13C or 15N specific labeling or unlabeling. Using as example the ABC transporter BmrA, a membrane protein of 600 residues, we demonstrate how these strategies can be applied. Indeed, even though there is no size limit in solid-state NMR, large (membrane) proteins are still a challenge due to heavy signal overlap. To initiate resonance assignment in these large systems, we describe how selectively labeled samples can be obtained with the addition of labeled or unlabeled amino acids in the medium. The reduced spectral overlap enabled us to identify typical spectral fingerprints and to initiate sequential assignment using the more sensitive 2D DARR experiments with long mixing time showing inter-residue correlations.

Direct amide 15N to 13C transfers for solid-state assignment experiments in deuterated proteins.

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only 1H-15N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than 1H-13C CP transfers, and employs a dipolar version of the INEPT experiment for N-C transfer. By avoiding HN-C (HN stands for amide protons) and C-C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired 13C spins.

CONFINE-MAS: a magic-angle spinning NMR probe that confines the sample in case of a rotor explosion.

Magic-angle spinning (MAS) is mandatory in solid-state NMR experiments to achieve resolved spectra. In rare cases, instabilities in the rotation or damage of either the rotor or the rotor cap can lead to a so called "rotor crash" involving a disintegration of the sample container and possibly the release of an aerosol or of dust. We present a modified design of a 3.2 mm probe with a confining chamber which in case of a rotor crash prevents the release of aerosols and possibly hazardous materials. 1D and 2D NMR experiments show that such a hazardous material-confining MAS probe ("CONFINE-MAS" probe) has a similar sensitivity compared to a standard probe and performs equally well in terms of spinning stability. We illustrate the CONFINE-MAS probe properties and performance by application to a fungal amyloid.

Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

The hepatitis B virus (HBV) icosahedral nucleocapsid is assembled from 240 chemically identical core protein molecules and, structurally, comprises four groups of symmetrically nonequivalent subunits. We show here that this asymmetry is reflected in solid-state NMR spectra of the capsids, in which peak splitting is observed for a subset of residues. We compare this information to dihedral angle variations from available 3D structures and also to computational predictions of "dynamic" domains and molecular hinges. We find that although, at the given resolution, dihedral angles variations directly obtained from the X-ray structures are not precise enough to be interpreted, the chemical-shift information from NMR correlates, and interestingly goes beyond, information from bioinformatics approaches. Our study reveals the high sensitivity with which NMR can detect the residues allowing the subtle conformational adaptations needed in lattice formation. Our findings are important for understanding the formation and modulation of protein assemblies in general.